Transcriptional and epigenetic changes associated with lead exposure in spermatozoa

Abstract

Lead (Pb) exposure is a risk factor of male infertility, while the transcriptional and epigenetic changes associated with lead exposure in spermatozoa are poorly understood. Our previous findings revealed significant changes in DNA methylation of the calcium (Ca) homeostasis pathway of human spermatozoa in men with a blood Pb level over 5 µg dl⁻¹, which was associated with decreased sperm motility. In this study, we explored the effects of Pb exposure on expression of differentially methylated genes (DMGs) by analyzing semen samples from six healthy, non-smoking, and non-drinking men (aged 20–40 years). Using methylated DNA immunoprecipitation sequencing (MeDIP-seq) and RNA sequencing (RNA-seq), we compared DNA methylation and RNA abundance patterns between two groups: three men with blood Pb level 0–2.5 µg dl⁻¹ and three men with blood Pb level 5–10 µg dl⁻¹. Additionally, we experimentally validated the regulatory function of the differentially methylated regions associated with 11 hub genes using dual-luciferase reporter assays. We revealed differences in promoter activity between methylated and unmethylated promoter regions of seven cloned genes, namely calcium voltage-gated channel subunit alpha1 H (CACNA1H), calcium voltage-gated channel subunit alpha1 G (CACNA1G), calcium voltage-gated channel subunit alpha1 I (CACNA1I), calcium/calmodulin dependent protein kinase II gamma (CAMK2G), ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 3 (ATP2A3), solute carrier family 8 member A2 (SLC8A2), and glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D). Our results of Pb exposure-induced expression changes of essential genes associated with the calcium signaling pathway, particularly CACNA1H, SLC8A2, and GRIN2D, in spermatozoa, may be a potential cause of low sperm quality.

INTRODUCTION

Lead (Pb) is a common toxic heavy metal widely used in gasoline, water supply pipelines, ceramic glazing, Pb-based paints, and manufacturing industries.1,2,3 It gradually accumulates in multiple tissues and, upon chronic exposure, causes harmful effects on different organs such as the nervous, neuroendocrine, and reproductive systems.4,5,6,7,8,9,10 Infertility is a state of reproductive system defined as the failure to achieve clinical pregnancy for more than 1 year in spite of regular and unprotected sexual behavior,11 of which male factor infertility accounts for 50%.12 Pb-induced male infertility includes the alteration of the neuro-endocrine hypothalamic-pituitary axis (HPA)13,14 and the impairment of spermatogenesis, sperm quality and function15,16,17,18,19 due to testicular damage.13

Pb exposure deleteriously affects sperm quality in a dose-dependent manner.20 The relationship between blood Pb level (BLL) and sperm quality has been well demonstrated, particularly at a concentration over 10 µg dl⁻¹, a threshold recognized as a public health concern by the Centers for Disease Control and Prevention (CDC).21 Low-dose Pb exposure in men with an average BLL below 10 µg dl⁻¹ has been demonstrated to be associated with reduced sperm quality, with limitation in inaccuracy caused by inclusion of individuals with a high BLL.22,23 For more precision, a population-based study was performed with exclusion of high-BLL individuals, indicating that low Pb exposure-induced aberrant sperm DNA methylation may cause decreased sperm quality.24

In this study, we performed transcriptome sequencing and double luciferase reporter gene experiment to investigate whether RNA expression of calcium (Ca) homeostasis pathway-associated genes is influenced by Pb exposure-induced differential DNA methylation, on the basis of our previous results showing that Pb exposure (BLL >5 µg dl⁻¹) significantly alters DNA methylation of the Ca homeostasis pathway of a spermatozoon.24

PARTICIPANTS AND METHODS

Study population

The study was approved by the Ethics Committee and Institutional Review Board of Shanghai Institute of Planned Parenthood Research (Shanghai, China; Approval No. PJ2018-08). All the study participants provided written informed consent.

To ensure the study cohort was well-defined and representative of the individuals potentially affected by Pb exposure, stringent inclusion and exclusion criteria were established. Inclusion criteria are as follows: (1) age between 20 years and 40 years; (2) no history of smoking or alcohol consumption; (3) blood cadmium (Cd) concentration < 1 µg dl⁻¹; (4) bisphenol A (BPA), di(2-ethylhexyl) phthalate (DEHP), mono(2-ethylhexyl) phthalate (MEHP), and dibutyl phthalate (DBP) concentrations <1 µg per µmol Cr; (5) normal testicular volume (≥12 ml for both left and right testes); (6) sperm density ≥1 × 10₆ ml⁻¹; and (7) absence of abnormal semen agglutination. Study population exclusion criteria are as follows: (1) sperm concentration < 1 × 10₆ ml⁻¹; (2) abnormal semen agglutination; or (3) testicular volume <12 ml (for either testis).

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Sperm analysis

Computer-assisted sperm analysis (CASA; WLJY-9000; Weili, Beijing, China) was utilized to evaluate sperm concentration, overall motility, and progressive motility. Sperm viability was assessed using vital staining with eosin–nigrosin dye. Manual evaluations conducted according to the World Health Organization (WHO) 5^th edition guidelines25 encompassed sperm morphology assessment and confirmation of CASA findings. All examinations were conducted by two qualified technicians (XMW and BW) who had undergone WHO-recommended certification and engaged in external quality control programs. When replicates for motility and sperm number in the counting chambers were within the acceptable differences specified by the WHO 5^th edition guidelines,25 the mean values were used for the final analysis.

Analysis of heavy metals and other chemicals

Blood Pb and Cd concentrations were determined using a graphite furnace atomic absorption spectrophotometer (Beijing Bohui Innovation Biotechnology Group Co., Ltd., Beijing, China). The labeling curve was drawn by using the standard solution of Pb and Cd provided by China National Research Center for Labeling Substances.24

Methylated DNA immunoprecipitation sequencing

Abstinence from sex for 2–7 days was required for all study participants before their provision of semen samples, which were collected by masturbation into 25-ml sterile polystyrene jars and analyzed within 60 min of ejaculation. CASA was performed to obtain sperm concentration, motility, and viability according to the WHO 5^th edition guidelines.25 Sperm DNA of six men aged 20–40 years without a history of smoking or drinking (three men with blood Pb level of 0–2.5 µg dl⁻¹ and three men with blood Pb level of 5–10 µg dl⁻¹; Supplementary Table 1) were extracted by DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and submitted for methylated DNA immunoprecipitation sequencing (MeDIP-seq). Genome integrity was assessed by agarose gel electrophoresis, DNA purity (optical density 260 nm/280 nm ratio) was measured by Nano drop (Thermo Fisher Scientific, Waltham, MA, USA), and DNA concentration was measured using Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Extracted DNA was fragmented through a Covaris sonication system (Covaris, Woburn, MA, USA), from which sequencing libraries were prepared on 5-μg DNA fragments. Steps of end repair, A-base addition, and adaptor ligation were performed with Illumina’s Single-End DNA sample preparation kit (Illumina, San Diego, CA, USA). Adaptor-ligated DNA was immunoprecipitated by anti-5-methylcytosine, followed by the purification and application for 50-bp single-end sequencing on an Illumina Hiseq2500 platform. The flowchart of sequencing and analysis process is shown in Figure 1.

Supplementary Table 1.

Clinical characteristics of study participants

	Groups (n)	All participants mean (s.d.)	Low-Pb-exposure group mean (s.d.)	High-Pb-exposure group mean (s.d.)
Statics		6	3	3
Age	6	33.67 (7.80)	32.33 (5.79)	35.00 (9.20)
Blood Pb (µg/dl)	6	3.98 (2.27)	1.79 (0.28)	6.17 (0.75)
Rapid progressive motility (%)	6	27.05 (7.27)	20.43 (3.17)	33.67 (2.87)
Slow progressive motility (%)	6	15.50 (5.88)	19.67 (5.25)	11.33 (2.63)
Non-progressive motility (%)	6	14.78 (9.84)	17.77 (13.16)	11.80 (1.66)
Sperm concentration (10−6/ml)	6	60.22 (35.95)	69.40 (46.62)	51.03 (15.60)
Sperm viability (%)	6	52.00 (6.23)	49.00 (2.16)	54.33 (6.85)
FSH (IU/l)	6	4.98 (1.26)	5.29 (0.96)	4.67 (1.43)
LH (U/l)	6	6.56 (2.67)	7.08 (3.26)	6.03 (1.76)
Testosterone (ng/ml)	6	5.15 (1.78)	5.23 (1.78)	5.07 (1.78)
Zn in semen (mmol/l)	6	2291.17 (783.65)	2143.95 (47.11)	2389.32 (998.97)
Ca in semen (mmol/l)	6	7.53 (1.43)	7.75 (1.51)	7.38 (1.36)
Mg in semen (mmol/l)	6	3.75 (1.50)	3.53 (0.11)	3.89 (1.92)

FSH: follicle-stimulating hormone; LH: luteinizing hormone; s.d.: standard deviation

Figure 1.

Flowchart of sequencing and analysis process of semen samples. BLL: blood lead level; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: Gene Set Enrichment Analysis; MeDIP: methylated DNA immunoprecipitation; IPA: Ingenuity Pathway Analysis.

RNA sequencing

Sperm RNA isolation was performed using RNeasy Mini Kit (Qiagen) added with a final concentration of 10 mmol l⁻¹ dithiothreitol (DTT). The extracted RNA was subjected to quality control assays, including RNA quantification with Qubit™ RNA HS Assay kit (Invitrogen, Carlsbad, CA, USA) and RNA integrity assessment with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA was sequenced on the Illumina Nova-seq platform (Illumina) with the generation of approximately 20 million reads per sample RNA-seq data after the construction of sequencing libraries with SMARTer® Universal Low Input RNA Kit (Takara, Shiga, Japan). Calculation of transcript per million (TPMs) and estimation of differential gene expression were performed using Kallisto-Sleuth pipelines.26

Availability of sequencing data

The raw sequence data from this study have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA002322 and HRA002316) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

Bioinformatic analysis

All methylation sequencing data passed quality checks for base composition through FastQC version 0.10.0.27 About 20 million reads were generated for each individual and mapped onto hg19 using Burrows-Wheeler Aligner (BWA).28 Peak calling, the computational identification of genomic regions with significantly enriched sequencing reads indicating DNA methylation sites, was performed using the MACS2 tool for both MeDIP-seq and chromatin immunoprecipitation sequencing (ChIP-seq) data.29 Before peak calling, PCR duplicate reads were removed to minimize the impact of technical replication on the results. Subsequently, the callpeak function of macs2 was used for peak analysis. The parameters included input file format (binary alignment/map [BAM]), peak type (broad peak), broad peak significance threshold (0.1), sequencing fragment extension length (300 bp), and bimodal mode processing (enabled [--fix-bimodal]). In order to evaluate the sample quality, R package ChIP-seq and Diffbind package were used in the evaluation of the sample quality and differential methylation regions (DMRs) between samples, respectively.30,31 Screening threshold for DMRs was log₂(fold change) >1 and false discovery rate (FDR) <0.05. Differential binding region was annotated by CHIPseeker.32

The plot of chromosome distribution of differentially methylated regions was implemented by R package chromoMap.33 Annotation of DMRs in promoter region and analyses of Gene Ontology (GO),34 Kyoto Encyclopedia of Genes and Genomes (KEGG),35 and Ingenuity Pathway Analysis (IPA) were performed by R package clusterprofiler.36 Heatmaps were drawn with genes where differential promoter regions were located using pheatmap package.

Total RNA sequencing reads were evaluated for quality control using FastQC software.27 Low-quality reads and sequencing adaptors were trimmed with Trimmomatic.37 Filtered reads were mapped to reference genome using HISAT2 version 2.1.0.38 DESeq2 was applied to the identification of differentially expressed genes (DEGs), with screening threshold for DEGs set to abs(log₂FC) >0, q-val< 0.05 (wherein, abs[log₂FC] refers to the absolute value of the log₂[fold change] in gene expression between conditions, and q-value is the adjusted P-value after correcting for multiple testing, typically used to control the FDR). R package pheatmap, topGO,39 and clusterProfiler36 were used for analyses of hierarchical clustering, GO,34 KEGG,35 and Gene Set Enrichment Analysis (GSEA), respectively.

Double luciferase reporter gene experiment

Promoter regions of 11 hub genes of interest were amplified and cloned upstream into firefly luciferase gene in pGL4.13 plasmid without SV40 promoter. A total of 5 × 10⁴ 293FT cells were seeded 24 h in advance to a 24-well plate, followed by the transfer of 200 µg CpG methyltransferase (M.SssI DNA Methyltransferase; New England Biolabs, Frankfurt, Germany) treated or untreated plasmids using Lipofectamine 2000 transfection reagent (Invitrogen). Forty-eight hours after transfection, luciferase activity was detected by a double luciferase reporter gene detection kit (Beyotime, Shanghai, China).

Statistical analyses

P < 0.05 was considered statistically significant. All statistical analyses were performed using R 3.6.3 package (https://rpkgs.datanovia.com; last accessed on 2021 January 15).

RESULTS

Distribution of differential DNA methylation

The chromosome distribution of DMRs between DNA methylation of spermatozoa from men with BLL of 0–2.5 µg dl⁻¹ and that of men with BLL of 5–10 µg dl⁻¹ was identified using MACS2 (Figure 2a). For differentially methylated genes (DMGs), we measured methylation levels of different gene regions because of unclear functions of DNA methylation of different regions of a specific gene. As Figure 2b shows, the number of hypomethylated genes was higher than that of hypermethylated genes in each region, and over 2000 hypomethylated genes were found to be located at body or promoter region. There were more than 1000 hypomethylated genes located at 3’ untranslated region (3’UTR), intron and intergenic region, while the number of hypomethylated genes located at 5’ untranslated region (5’UTR), 1^st expressed region (exon) and downstream region was smaller.

Figure 2.

(a) Chromosome distribution of differentially methylated regions (DMRs). The plot displays the distribution of differentially methylated regions at 22 autosomes, the X chromosome, and the Y chromosome. The region in blue is a hypomethylated region, and the region in yellow is a hypermethylated region. The value represents the log₂FC between DNA methylation of sperm from three men with BLL of 0–2.5 µg dl⁻¹ and that from three men with BLL of 5–10 µg dl⁻¹. (b) Barplot of differentially methylated genes (DMGs) in different regions. The x-axis labels different gene regions, including 5’ untranslated region (5’UTR), 1^st expressed region (exon), body, 3’ untranslated region (3’UTR), intergenic region, promoter region, intron, and downstream region. 5’UTR refers to the 5’ untranslated region located between the transcriptional start site (TSS) and ATG start site. 1^st exon is short for the first exon of a gene. Body is the region located between ATG start site and stop codon. 3’UTR refers to 3’ untranslated region located between stop codon and poly-A tail. Intergenic is short for DNA sequences located between genes. Promoter stands for DNA sequences that RNA polymerase recognizes, binds to, and initiates transcription. Intron means a stretch of DNA sequence that is not expressed in a gene product. Downstream is short for downstream sequences. The y-axis is the number of DMGs. Green color represents hypermethylated genes. Orange color represents hypomethylated genes. FC: fold change

Transcriptome sequencing of sperm RNA of men exposed to Pb

In transcriptome sequencing results, there were 1579 DEGs, of which 1150 were significantly upregulated and 429 were significantly downregulated in sperm RNA from men with BLL of 5–10 µg dl⁻¹ (Figure 3a and Supplementary Table 2). Upregulated genes accounted for the majority of all DEGs, accompanied with hypomethylated gene corresponding to highly expressed genes (Figure 3b). For identified DEGs, GO and KEGG were performed with clusterProfiler.30,36 GO analysis showed that DEGs were mainly enriched in pathways determining sperm cytoskeleton and motility (e.g., cilium assembly, cilium organization, and microtubule organizing) as well as RNA metabolism (e.g., non-coding RNA metabolic process, RNA catabolic process, and mRNA catabolic process), as shown in Figure 3c, while only two pathways, RNA transport (hsa03013) and RNA degradation (hsa03018), were obtained in KEGG analysis (data not shown). Apart from GO and KEGG assays, which are sensitive to threshold setting for DEGs, we also analyzed GSEA, requiring no thresholds. Expression levels of two gene sets (calcium ion transport and calcium signaling pathway), sets with significant methylation changes in promoter regions, were also significantly changed in GSEA results (normalized enrichment score [NES] >1, FDR < 0.25; Figure 3d).

Figure 3.

Transcriptome sequencing (RNA-seq) analysis results. (a) Volcano plot of gene expression. High_up represents genes with significantly upregulated expression. Low_up represents genes with significantly downregulated expression. (b) Bubble plot of GO analysis of differentially expressed genes (DEGs). (c) Barplot of four groups overlapping in each region. Hyper_down represents hypermethylated and downregulated genes. Hyper_up represents hypermethylated and upregulated genes. Hypo_down represents hypomethylated and downregulated genes. Hypo_up represents hypomethylated and upregulated genes. (d) GSEA profiles depicting two significant enrichment sets. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: Gene Set Enrichment Analysis; 5’UTR: 5’ untranslated region; 3’UTR: 3’ untranslated region; NES: normalized enrichment score; ncRNA: non-coding RNA; BP: biological process.

Supplementary Table 2.

Clinical characteristics of study participants

	RL-1	RL-2	RL-3	RH-1	RH-2	RH-3
Age	32	30	22	35	28	26
Blood Pb (µg/dl)	23.84	20.49	18.69	82.14	81.29	70.20
Blood Cd (µg/dl)	0.19	0.94	0.69	0.088	0.74	0.61
Rapid progressive motility (%)	21.00	16.30	24.00	34.00	30.00	37.00
Slow progressive motility (%)	15.00	27.00	17.00	15.00	9.00	10.00
Non-progressive motility (%)	7.00	36.30	10.00	14.00	10.00	11.40
Sperm concentration (10−6/ml)	40.00	135.20	33.00	40.00	40.00	73.10
Sperm volume (ml)	4	2	1.5	4	2	1.5
Zn in semen (mmol/l)	2715.77	2096.84	2191.05	3307.21	2860.46	1000.31
Ca in semen (mmol/l)	8.56	6.25	9.26	8.81	7.78	5.56
Mg in semen (mmol/l)	4.87	3.42	3.65	6.21	3.97	1.51
Zn in urine (mmol/l)	7.83	8.82	7.65	6.26	6.31	1.97
Ca in urine (mmol/l)	0.49	4.99	3.59	1.56	2.58	2.25
Mg in urine (mmol/l)	1.05	2.07	2.57	1.51	1.29	1.83
BPA (µg/µgmolcr)	0.89	0.038	0.29	0.16	0	0
DEHP (µg/µgmolcr)	0.044	0	0.38	0.034	0.99	0.20
DBP (µg/µgmolcr)	0.49	0.24	0.21	0.10	0.19	0.26
MEHP (µg/µgmolcr)	0.88	0	0	0	0	0

Zn: zinc; Ca: calcium; Mg: magnesium; BPA: bisphenol A; DEHP: di (2-ethylhexyl) phthalate; DBP: dibutyl phthalate; MEHP: mono (2-ethylhexyl) phthalate

Differences in promoter activity between methylated and unmethylated promoter regions

To investigate the functions of promoter regions of DMGs and the effects of methylation, we performed dual luciferase reporter assays (Figure 4a). A total of seven promoter regions (calcium voltage-gated channel subunit alpha1 H [CACNA1H], calcium voltage-gated channel subunit alpha1 G [CACNA1G], calcium voltage-gated channel subunit alpha1 I [CACNA1I], calcium/calmodulin dependent protein kinase II gamma [CAMK2G], ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 3 [ATP2A3], solute carrier family 8 member A2 [SLC8A2], and glutamate ionotropic receptor NMDA type subunit 2D [GRIN2D]) were cloned upstream of the firefly luciferase gene in pGL4.13-Neg (pGL4.13 plasmid without SV40 promoter), while the remaining four regions could not be cloned, likely owing to high GC content in their promoter sequences. In comparison with the positive control (normal pGL4.13 plasmid), the firefly luciferase activity was reduced 10 times in cells transfected with pGL4.13-Neg. Promoters of CACNA1G (P < 0.01), SLC8A2 (P < 0.01) and GRIN2D (P < 0.01) significantly increased firefly luciferase activity compared with the negative control, however, luciferase activity in samples of CACNA1H, CACNA1I, and ATP2A3 promoters did not increase significantly (all P > 0.05; Figure 4b). By contrast, luciferase activity driven by CAMK2G promoter was significantly lower than that of pGL4.13-Neg (Figure 4b).

Figure 4.

(a) Heatmap summarizing significantly differentially methylated genes enriched in the calcium homeostasis pathway. (b) Effects of different promoters on expression of firefly luciferase. RL group: BLL 0–2.5 µg dl⁻¹; RH group: BLL 5–10 µg dl⁻¹. NS: not significant; ^**P < 0.01; ^***P < 0.001. BLL: blood lead level; GRIN2D: glutamate ionotropic receptor NMDA type subunit 2D; CACNA1I: calcium voltage-gated channel subunit alpha1 I; CACNA1H: calcium voltage-gated channel subunit alpha1 H; CACNA1G: calcium voltage-gated channel subunit alpha1 G; CAMK2G: calcium/calmodulin dependent protein kinase II gamma; ATP2A3: ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 3; SLC8A2: solute carrier family 8 member A2; RYR1: ryanodine receptor 1; PRKACA: protein kinase cAMP-activated catalytic subunit alpha; ATP2A1: ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 1; NEG: negative; PRF-con: positive regulatory factor control.

Comparing the abilities of methylated and unmethylated promoter regions of initiating luciferase transcription, the results showed that promoter activities of CACNA1H, SLC8A2, and GRIN2D were significantly decreased (all P < 0.05) after methylation, but the decreased promoter activities of CAMK2G and ATP2A3 showed insignificant level (both P > 0.05). On the contrary, CACNA1I increased significantly after methylation (P < 0.05; Figure 5).

Figure 5.

Detection of promoter function by double luciferase reporter gene assay. Changes of expression ability of firefly luciferase activated by different promoters before and after methylation. NS: not significant; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. GRIN2D: glutamate ionotropic receptor NMDA type subunit 2D; CACNA1I: calcium voltage-gated channel subunit alpha1 I; CACNA1H: calcium voltage-gated channel subunit alpha1 H; CACNA1G: calcium voltage-gated channel subunit alpha1 G; CAMK2G: calcium/calmodulin dependent protein kinase II gamma; ATP2A3: ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 3; SLC8A2: solute carrier family 8 member A2; Met: methylation; Neg: negative control.

In summary, promoters of CACNA1H, SLC8A2, and GRIN2D were significantly less efficient after methylation. More repetitions are needed to determine the effects of methylation on promoter regions of CAMK2G and ATP2A3, with decreasing trends after methylation and P values ranging from 0.05 to 0.1.

DISCUSSION

Our previous findings indicated that Pb exposure (BLL <10 µg dl⁻¹) was associated with reduced sperm motility in adult men, which is inconsistent with results of previous studies suggesting low blood Pb exposure is uncorrelated with semen quality parameters (volume and pH) or sperm quality markers (concentration, motility, morphology, and viability),40 and lowers sperm concentration and movement.10 This discrepancy may reflect the lack of assessing other risk factors (e.g., levels of trace elements and plastifiers) and inclusion of individuals with BLL over 10 µg dl⁻¹ in these studies. Pb exposure may cause epigenetic changes in human spermatozoa, specifically affecting DNA methylation patterns, which is consistent with previous studies reporting correlations between Pb exposure and altered DNA methylation.24,41 However, no global differences were observed in DNA hydroxymethylation of spermatozoa, suggesting that DNA methylation and hydroxymethylation may respond differently to Pb exposure. Hydroxymethylation may exhibit lower sensitivity or requiring different exposure conditions to manifest detectable changes.

The current study particularly explored the Pb exposure-induced differential RNA expression of genes associated with Ca homeostasis pathway in spermatozoa. Through RNA sequencing and functional enrichment analysis of DEGs, we found that previously observed hypomethylation did change expression patterns of Ca signaling pathway-associated genes. Enrichment results of DMGs supported these findings, highlighting pathways related to sperm motility, cytoskeleton (important for sperm fertilization ability),42 RNA metabolism, and calcium signaling. Dual-luciferase reporter assays further revealed differences in promoter activity between methylated and unmethylated promoter regions of DMGs of interest. Gene products encoded by CAMK2G, SLC8A2, and ATP2A3 were predicted to be related to biological processes such as synapse plasticity, catalysis of ATP hydrolysis coupled with calcium translocation, regulation of postsynaptic cytosolic calcium ion concentration, and calcium antiporter activity.43,44,45,46,47 CACNA1G and CACNA1H encode the gene products calcium voltage-gated channel subunit alpha1 G and calcium voltage-gated channel subunit alpha1 H, respectively. These subunits are complex members of voltage-sensitive calcium channels, which mediate the influx of calcium ions into excitable cells. They are also involved in various calcium-dependent processes, including cell death, cell division, cell motility, gene expression, hormone/neurotransmitter release, and muscle contraction.48,49 Differential expression of CACNA1G has been demonstrated to be associated with chicken sperm storage duration,50 and the strong correlation between CACNA1H and sperm morphology and differential gene expression is recognized as a novel biomarker that can be used for sperm analysis and the exploration of male infertility.51 Thus, we hypothesize that Pb exposure-related changes in the expression of sperm genes involved in calcium signaling may be a cause of decreased sperm quality. This is particularly significant since calcium plays essential roles in sperm motility,52,53 and modifications of calcium homeostasis-related pathways are an important mechanism through which environmental exposures can affect human health.54

Several calcium signaling genes identified as affected by Pb exposure in spermatozoa, particularly CACNA1H, have previously been implicated in pituitary hormone secretion regulation.55 Several calcium signaling genes identified as affected by Pb exposure in spermatozoa, particularly CACNA1H, have previously been implicated in pituitary hormone secretion regulation.55 This raises the important question of whether environmental Pb exposure that damages sperm CACNA1H expression simultaneously disrupts the hypothalamic-pituitary-gonadal (HPG) axis, and whether these Pb induced epigenetic modifications in spermatozoa can be transmitted to offspring, with the altered gene expression indicating broader calcium signaling disruption that affect the offspring’s HPG axis development and function.

Our study provides insights into the transcriptional and epigenetic changes associated with Pb exposure in spermatozoa, particularly in relation to Ca signaling. However, we acknowledge several limitations to be considered when interpreting our findings. First, the sample size of our study was small, which may limit the generalizability of our findings. Future studies with larger sample sizes are needed to confirm and extend our results. Second, there were baseline imbalances such as age in our study population, which may have influenced our results. In future studies, we will consider adjusting for these potential confounding factors to ensure more reliable data. Third, our study was conducted in a Chinese population, possibly limiting the applicability of our findings to other ethnic or racial groups. Further studies in diverse populations are required to determine whether our findings are universal. Fourth, our inclusion criteria did not account for body mass index (BMI), which could influence sperm quality and epigenetic modifications, and future studies should consider incorporating BMI as a factor to control these potential confounding effects. Fifth, although blood Pb levels were measured here, seminal Pb levels may provide additional insights into direct reproductive toxicity, and future studies incorporating both indicators are warranted to refine mechanistic understanding.

Despite these limitations, our study has important implications for clinical practice and future research. Our findings suggest that assessing the methylation status or expression profiles of related sperm genes, in conjunction with measuring Pb levels in blood (and potentially in seminal plasma for more direct assessment of reproductive tract exposure), could guide clinical decisions in assisted reproductive therapy. For patients with high Pb levels, chelation therapy could be considered to reduce lead levels and potentially improve sperm quality. The biomarkers identified in our study could serve as indicators of the efficacy of chelation therapy in men with elevated lead exposure who are planning to father children or undergoing assisted reproductive therapy.

In conclusion, our study indicates that Pb exposure may lead to changes in the expression of essential genes associated with Ca signaling pathway in spermatozoa, contributing to decreased sperm quality. Further studies are needed to confirm these findings and to explore potential underlying mechanisms.

CONCLUSIONS

Our study demonstrated that Pb exposure (BLL <10 µg dl⁻¹) is associated with differential DNA methylation and an altered global transcriptome profile in human spermatozoa, particularly affecting genes in the Ca homeostasis pathway. Hypomethylation changed the expression patterns of Ca signaling pathway-associated genes, with functional enrichment in pathways critical for sperm motility and fertilization ability. Significant differences in promoter activity between methylated and unmethylated regions in seven key genes were identified. These findings suggest that Pb exposure-induced changes in Ca signaling pathway genes may contribute to poor sperm quality.

AUTHOR CONTRIBUTIONS

TCZ takes primary responsibility for the paper. XZ and XRS collected the data. XZ, XRS, and BW performed the statistical analysis. XMW participated in the data analysis. HJS drafted the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.